Oligodeoxynucleotides, which are complementary to certain gene messages or viral sequences, are referred to as "anti-sense" compounds. These compounds have also been reported to have inhibitory effects against Rous sarcoma virus and human T-cell lymphotropic virus type III (HTLV-III), now called Human Immunodeficiency Virus (HIV). However, the susceptibility of the phosphodiester linkage in normal oligodeoxynucleotides to degradation by nucleases would be expected to reduce their potency and in vivo persistence as anti-viral agents.
Methylphosphonate-oligodeoxynucleotide analogs are resistant to nucleases, and because they are uncharged have increased hydrophobicity, which reportedly confers increased cell membrane permeability upon these compounds. The methylphosphonate-oligodeoxynucleotides have been found to exhibit antiviral activity, but these compounds may require high concentrations, typically 100-300 micromoles, in order to elicit strong antiviral effects.
A number of investigators have studied the inhibitory properties of both normal oligodeoxynucleotides and analogs of oligodeoxynucleotides. T'so and coworkers evaluated ethyl phosphotriester and methylphosphonate analogs of oligodeoxynucleotides as nonionic compounds that penetrate cells, and are relatively resistant to degradation by nucleases (cf. U.S. Pat. No. 4,469,863). The ethyl compounds were found, however, to have the disadvantage of undergoing degradative de-ethylation in cells. The methylphosphonates were found to be more stable and to have antiviral activity.
The methylphosphonate analogs as described above have been said to inhibit expression of some genes. However, these compounds have a number of serious disadvantages:
(1) Such compounds are very sensitive to base-catalyzed hydrolysis, making them relatively difficult to synthesize on a routine basis, as compared to poly-anionic oligodeoxynucleotides; PA1 (2) The compounds have relatively low solubility in aqueous media, thus restricting their potential biological/chemotherapeutical usage, as compared to poly-anionic oligodeoxynucleotides; PA1 (3) Relatively high concentrations of these compounds appear to be required to elicit antiviral activity; and PA1 (4) There is poor hybridization because of the steric effect of the methyl group.
These factors taken together make chemotherapy impractical in humans with these compounds.
Early work by Zamecnik and co-workers used normal unmodified oligodeoxynucleotides, as well as 3'-end-blocked (2',3'-dideoxyribosyl) analogs of oligodeoxynucleotides, to inhibit the transforming ability, replication and translation of Rous sarcoma virus in vitro. This approach has been extended by both Zamecnik et al. in PNAS, U.S.A., 83:4143-4146 (1986), who studied the inhibition of HIV virus in cultured human cells, and Wickstrom et al., in J. Biochem, Biophys. Methods, 13:97-102 (1986), who investigated the inhibition of the translation of mRNA from vesicular stomatitus virus.
Compared to the aforementioned methylphosphonate analogs, the unmodified, or "O", oligodeoxynucleotides offer the advantages of cost-effectiveness and synthetic accessibility, and moreover appear to have the added advantage of lower effective dosages. However, these unmodified oligodeoxynucleotides are susceptible to degradation by nucleases, even with the inclusion of a 3'-end-blocking residue. Consequently, the use of these compounds in vitro is significantly restricted, and it is highly unlikely that they can be successful in vivo.
The finding that human T-cell lymphotropic virus type III (HTLV-III), hereinafter referred to as Human Immunodeficiency Virus (HIV), is the causative agent of acquired immune deficiency syndrome (AIDS), prompted considerable interest in the development of chemotherapeutic approaches to the treatment of AIDS. A variety of compounds have been reported to have in vitro activity against HIV, although none of these compounds is known to inhibit the expression of the integrated viral genome.
The phosphorothioate oligodeoxynucleotides of several sequences, including sense, anti-sense, nonsense, and homo-oligomers, have been found to inhibit HIV, so that the mechanism of inhibition was unclear. Subsequent work has indicated that the inhibition by homo-oligomers, not complementary to any known sequence in the HIV genome, results from interaction and interference with the function of reverse transcriptase of HIV at low concentrations, i.e. less than 10 micromoles. The mechanism initially expected for complementary base sequence inhibition, known as "translation arrest" of the corresponding mRNA, is apparently not operative in the retrovirus until much higher concentrations of the phosphorothioate compounds are reached, i.e., greater than 25 micromoles. This explains the lack of sequence specificity observed for this inhibitory process.
Attempts have previously been made to inhibit proliferation of normal lymphocytes and HL60 cells using a normal oligodeoxynucleotide (ODN) sequence complementary to a region near the initiation codon of the c-myc gene. Although these results were encouraging, they demonstrated that normal anti-sense c-myc ODNs, even at concentrations as high as 100 micromoles, are not suitable for prolonged inhibition of c-myc protein expression in HL60 cells, even though these normal oligodeoxynucleotides were capable of preventing normal lymphocyte proliferation and c-myc protein expression. It would appear that these insufficiencies of the normal oligodeoxynucleotides, lack of prolonged inhibitory effect and high concentrations required even for short-term effectiveness, were due to enzymatic hydrolysis during the course of the experiment.
Heikkila et al., in Nature, 328, Jul. 30 1987, pp. 445-449, disclose that a c-myc oligodexoynucleotide inhibits entry into the S phase in lymphocyte mitogenesis. Small antisense oligomers were added to bulk cell cultures. A pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc RNA inhibits mitogen-induced c-myc protein expression in human T-lymphocytes and prevents S phase entry.